The classical AM fungal identification method of spore extraction from soil and further spore morphological analysis  is fraught with complicating issues due to the various strategies and forms of AM fungi, e. This is especially true in the case of root colonization analyses, which can determine percentage of roots colonized by AM fungi.
The major problem with this analysis is in field soils, which contain multiple species of AM fungi in association with a target plant at the same time see Ecology of AM. The identification of the associated fungal symbionts is impossible without the use of molecular methods.
Though genetic analysis of AM fungal communities has advanced a great deal in the past decade, the methodology is not yet completely refined. Below is an overview of the methods used in molecular genetic analyses of AM fungi, along with applications to research, future directions and some of their problems. Genetic analyses of AM fungi from soil and root samples range in their applicability to answer ecological or phylogenetic questions.
DNA analyses utilize various nuclear markers to describe AM fungi and represent different regions of the nuclear ribosomal operon 18S rRNA found in all eukaryotic organisms. The DNA analysis of AM fungi using these markers began in the early s  and are continuing to be developed today.
The SSU region has been used most frequently in ecological studies,  while the ITS and LSU regions have been predominantly used in taxonomic constructions of the phylum Glomeromycota. For analysis of DNA, samples should either be processed immediately or kept frozen prior to nucleic acid extraction. There are many PCR conditions proposed for analysis of AM fungi and some of the most accessible are briefly summarized below.
One difficulty with the genetic analysis of arbuscular mycorrhizal fungi has been the selection of ideal, comprehensive, and repeatable primers or primer sets. These sequence markers are designed for the nuclear ribosomal RNA rRNA in the 18S region and are either used individually or in some combination.
Currently, there is no consensus as to which primers or primer sets, being used with varying degrees of success, repeatability and species-level resolution, are best for molecular genetic analysis of AMF. Additionally, the current advances and coming changes in genetic sequencing technology, e. Illumina MiSeq as opposed to pyrosequencing that is capable of these long read lengths. Though Roche Diagnostics has announced the discontinuation of the platform for ,  it is still commonly used in genetic analyses. Perhaps new 'all-inclusive' AM specific primers should be created to support the new technologies for as descriptive a molecular analysis from the "Kruger" primer set using pyrosqeuncing, as shown below.
The reverse may also be true, where molecular technologies should be developed with both long read lengths which would allow for large primer sets as well as sequencing depth. Kohout et al. Results of their experiment are summarized below.
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Fairly recent developments in qPCR markers allow researchers to explore the relative abundance of AM fungal species within roots in greenhouse experiments as well as in the field to identify local AM fungal communities. These AM specific primers discussed above can be chosen by the researcher and this decision is typically guided by the question at hand, resources available, and willingness to troubleshoot in the lab. DNA microarray analysis is currently being used in AM fungal research to simultaneously measure the expression of many genes from target species or experimental samples.
The most common tool or method is to use functional gene array FGA technology, a specialized microarray that contains probes for genes that are functionally important in microbial processes such as carbon, nitrogen or phosphorus cycling. FGAs have the ability to simultaneously examine many functional genes. Specific organismal chemical signatures can be used to detect biomass of more cryptic organisms, such as AM fungi or soil bacteria. Lipids, more specifically phospholipids and neutral lipids, contain fatty acids connected to a glycerol backbone.
The fatty acid composition of organisms varies, and the proportions of specific fatty acids can be organism specific. Energy is mainly stored in AM fungi as neutral lipids in storage structures like spores and vesicles.tarcsiadam.com/cache/virgo/naro-kamal-kapoor-monthly.php
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Because of this NLFA correlates quite well with the number of spores in a given volume of soil. Problems with lipid fatty acid analyses include the incomplete specificity of fatty acids to AM fungi, the species- or genera-specific variation in fatty acid composition can complicate analysis in systems with multiple AM fungal species e. An exciting prospect for future analysis of AM fungi is the use of stable isotope probes.
Stable isotope probing SIP is a technique that can be used to determine the active metabolic function of individual taxa within a complex system of microbes. This level of specificity, linking microbial function and phylogenetics, has not been achieved previously in microbial ecology. This method can also be used independently of classical culture methods in microbial ecology, allowing for in situ analysis of functional microbes.
The H 2 18 O, or heavy water method will target all organisms that are actively growing, and induce little influence on growth itself. This would be especially true with most greenhouse experiments with arbuscular mycorrhizas because plants must be watered anyway, and water does not directly select for organisms with specific metabolic pathways,  as would happen when using 13 C and 15 N. It seems that AM fungi have their unique features to have bacterial type core enzyme as well as the large number of Argonaute proteins in their sRNA processing system or RNAi system.
Disturbance of native plant communities in desertification -threatened areas is often followed by degradation of physical and biological soil properties, soil structure, nutrient availability, and organic matter. When restoring disturbed land, it is essential to replace not only the above ground vegetation but also biological and physical soil properties.
A relatively new approach to restoring land is to inoculate soil with AM fungi when reintroducing vegetation in ecological restoration projects phytoremediation. It has enabled host plants to establish themselves on degraded soil and improve soil quality and health. Soils' quality parameters were significantly improved long-term when a mixture of indigenous arbuscular mycorrhizal fungi species was introduced compared to noninoculated soil and soil inoculated with a single exotic species of AM fungi.
Many modern agronomic practices are disruptive to mycorrhizal symbiosis. There is great potential for low-input agriculture to manage the system in a way that promotes mycorrhizal symbiosis. Conventional agriculture practices, such as tillage , heavy fertilizers and fungicides , poor crop rotations, and selection for plants that survive these conditions, hinder the ability of plants to form symbiosis with arbuscular mycorrhizal fungi.
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Most agricultural crops can perform better and are more productive when well-colonized by AM fungi. AM symbiosis increases the phosphorus and micronutrient uptake and growth of their plant host George et al. Management of AM fungi is especially important for organic and low-input agriculture systems where soil phosphorus is, in general, low, although all agroecosystems can benefit by promoting arbuscular mycorrhizae establishment. Some crops that are poor at seeking out nutrients in the soil are very dependent on AM fungi for phosphorus uptake.
For example, flax , which has poor chemotaxic ability, is highly dependent on AM-mediated phosphorus uptake at low and intermediate soil phosphorus concentrations Thingstrup et al. Proper management of AMF in the agroecosystems can improve the quality of the soil and the productivity of the land. Agricultural practices such as reduced tillage, low phosphorus fertilizer usage, and perennialized cropping systems promote functional mycorrhizal symbiosis.
Tillage reduces the inoculation potential of the soil and the efficacy of mycorrhizaes by disrupting the extraradical hyphal network Miller et al. By breaking apart the soil macro structure, the hyphal network is rendered non-infective Miller et al. The disruption of the hyphal network decreases the absorptive abilities of the mycorrhizae because the surface area spanned by the hyphae is greatly reduced.
In reduced-tillage system, heavy phosphorus fertilizer input may not be required as compared to heavy-tillage systems. This is due to the increase in mycorrhizal network, which allows mycorrhizae to provide the plant with sufficient phosphorus Miller et al. The benefits of AMF are greatest in systems where inputs are low. Heavy usage of phosphorus fertilizer can inhibit mycorrhizal colonization and growth. As the soil's phosphorus levels available to the plants increases, the amount of phosphorus also increases in the plant's tissues, and carbon drain on the plant by the AM fungi symbiosis become non-beneficial to the plant Grant Cover crops are grown in the fall, winter, and spring, covering the soil during periods when it would commonly be left without a cover of growing plants.
Mycorrhizal cover crops can be used to improve the mycorrhizal inoculum potential and hyphal network Kabir and Koide , Boswell et al.
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Since AM fungi are biotrophic, they are dependent on plants for the growth of their hyphal networks. Growing a cover crop extends the time for AM growth into the autumn, winter, and spring. Promotion of hyphal growth creates a more extensive hyphal network. The mycorrhizal colonization increase found in cover crops systems may be largely attributed to an increase in the extraradical hyphal network that can colonize the roots of the new crop Boswell et al.
The extraradical mycelia are able to survive the winter, providing rapid spring colonization and early season symbiosis McGonigle and Miller This early symbiosis allows plants to tap into the well-established hyphal network and be supplied with adequate phosphorus nutrition during early growth, which greatly improves the crop yield.
Restoration of native AM fungi increases the success of ecological restoration project and the rapidity of soil recovery. It is defined by its extraction conditions and reaction with the antibody Mab32B There is other circumstantial evidence to show that glomalin is of AM fungal origin. When AM fungi are eliminated from soil through incubation of soil without host plants, the concentration of GRSP declines.
Suppression of the activity of arbuscular mycorrhizal fungi by the soil microbiota
A similar decline in GRSP has also been observed in incubated soils from forested, afforested, and agricultural land  and grasslands treated with fungicide. Glomalin is hypothesized to improve soil aggregate water stability and decrease soil erosion. A strong correlation has been found between GRSP and soil aggregate water stability in a wide variety of soils where organic material is the main binding agent, although the mechanism is not known.
From Wikipedia, the free encyclopedia. Main article: Phytoremediation. New Phytologist. Terrestrial plant ecology.
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- Crossing in Complexity: Interdisciplinary Application of Physics in Biological and Social Systems;
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Frontiers in Physics. Bibcode : Natur. Bibcode : PNAS Current Science. November Molecular Plant-Microbe Interactions. Introductory mycology, 4th ed.
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